In addition to its evolutionary optimized functions, the extraordinary physical and functional properties of nucleic acids provide the opportunity for a plethora of new bio-molecular devices and methods. Designer nucleic acids have been contemplated for therapeutic entities, biosensors, nano-scale devices and tools for molecular computation. The methods exploit the characteristics of DNA self-assembly, electro-conductivity, information elements, amplification, switching, molecular detection and catalytic activity. Further, since DNA is robust, stable and thermostable it provides an ideal material for molecular engineering of mechanical or computation devices.
Single stranded nucleic acids, such as DNA and RNA, have the ability to fold into complex three-dimensional structures that can function as highly specific receptors (aptamers) and catalysts (ribozymes and DNAzymes). Further, the requirement for complementarity between nucleic acid strands for hybridization forms the basis for a wide range of techniques, which allow target detection (e.g. microarray analysis, Northern blotting or Southern blotting), and/or target amplification (e.g. the polymerase chain reaction). Further, hybridization provides the basis for nucleic acid nano-scale construction and for DNA based computational strategies.
Self-replication is a process by which individuals can duplicate (copy) themselves. In such processes, the products of each reaction direct the formation of the new copies (replicons) of the individual from component parts. A wide variety of techniques have been developed for the self-replication of nucleic acid sequences.
Methods for in vitro replication of target nucleic acid sequences (target amplification) are well known in the art. Many of these methods require oligonucleotide primers, capable of specific hybridization with the target DNA or RNA, which can be extended by DNA or RNA polymerase to create a new copy of the target (an amplicon), using the target as a template to direct synthesis. Such techniques (reviewed Schweitzer and Kingsmore, 2001) include the polymerase chain reaction, strand displacement amplification, rolling circle amplification, and loop-mediated isothermal amplification, transcription-mediated amplification, self-sustained sequence replication and nucleic acid sequence replication based amplification. An alternative approach, known as the ligase chain reaction (“LCR”) uses a protein ligase to amplify nucleic acid targets. The reaction depends on the capacity of the ligation products from each round to serve as templates to direct the ligation of new copies of the target (Barany, 1991).
Target amplification technologies, such as those above, have been widely used in research and/or in clinical diagnostics. However, despite their power, each has inherent disadvantages. They all require the use of protein enzymes (e.g. DNA polymerase, RNA polymerase, reverse transcriptase, and or ligase). The inclusion of protein enzymes increases the complexity and cost of reagent manufacture and decreases the shelf life of kits containing reagents. Other associated technical challenges include contamination by replicons (target amplicons) from previous reactions leading to false positive signal, and/or background signal caused by replication of primer sequences (primer-dimers) or background caused by target-independent ligation.
A wide variety of nucleic acid molecules, with enzymatic or catalytic activity, have been discovered in the last 20 years. RNA enzymes (“ribozymes”) occur in nature but can be engineered to specifically recognize and modify a target RNA substrate (Haseloff and Gerlach, 1988). In vitro evolution techniques have facilitated the discovery and development of many more catalytic nucleic acids, including deoxyribonucleic acids often referred to as “DNA enzymes” or “DNAzymes” (reviewed Emilsson and Breaker, 2002). In vitro evolved DNAzymes and/or ribozymes have been discovered which have the capacity to catalyse a broad range of reactions including cleavage of nucleic acids (Carmi et al., 1996; Raillard and Joyce, 1996; Breaker, 1997; Santoro and Joyce, 1998), ligation of nucleic acids (Cuenoud and Szostak, 1995, Prior et al., 2004), porphyrin metallation (Li and Sen, 1996), and formation of carbon-carbon bonds (Tarasow et al., 1997), ester bonds (Illangasekare et al., 1995) or amide bonds (Lohse and Szostak, 1996).
In particular, DNAzymes and ribozymes have been characterized which specifically cleave distinct nucleic acid sequences after hybridizing via Watson Crick base pairing. DNAzymes are capable of cleaving either RNA (Breaker and Joyce, 1994; Santoro and Joyce, 1997) or DNA (Carmi et al., 1996) molecules. Ribozymes are also able to cleave both RNA (Haseloff and Gerlach, 1988) and DNA (Raillard and Joyce, 1996) target sequences. The rate of catalytic cleavage of many nucleic acid enzymes is dependent on the presence and concentration of divalent metal ions such as Ba2+, Sr2+, Mg2+, Ca2+, Ni2+, Co2+, Mn2+, Zn2+, and Pb2+ (Santoro and Joyce, 1998; Brown et al., 2003).
The “10:23” and “8:17” DNAzymes are capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds to create reaction products which have 2′,3′-cyclic phosphate and 5′-hydroxyl groups (Santoro and Joyce, 1997; reviewed Emilsson and Breaker, 2002). Examples of deoxyribozymes (DNAzymes), which can ligate 2′,3′-cyclic phosphate and 5′-hydroxyl products include the “7Z81” and “7Z48” ligases (Prior, 2004).
Several catalytic nucleic acids, including the hammerhead ribozyme, the 10:23 and 8:17 DNAzymes, and the “7Z81” and “7Z48” ligases have similar basic structures with multiple domains. These nucleic acid enzymes have a conserved catalytic domain (catalytic core) flanked by two non-conserved substrate-binding domains (“arms”), which specifically recognize and hybridise to the substrate. While these nucleic acid enzymes can function as true multiple turnover enzymes, each enzyme only has the capacity to recognise one molecule, namely the substrate which it can then catalytically modify.
Catalytic nucleic acids have been shown to tolerate only certain modifications in the area that forms the catalytic core (Perreault et al., 1990; Perreault et al., 1991; Zaborowska et al., 2002; Cruz et al., 2004; Silverman, 2004)). Depending on the stringency of the reaction conditions, some degree of mismatch may be tolerated within the substrate arms. However, the requirement for Watson Crick base pairing is sufficiently strict so as to have enabled the development of protocols that use catalytic nucleic acids to facilitate the discrimination of closely related sequences (Cairns et al., 2000) (WO 99/50452).
“Aptamers” are DNA, RNA or peptide sequences that have the ability to recognize one or more ligands with great affinity and specificity due to their high level structure, for example, a 3-D binding domain or pocket. Many aptamers have been evolved in vitro for their capacity to bind to ligands, including for example, nucleic acids, proteins, prions, small organic compounds, and/or entire organisms. “Aptazymes” have sequences comprised of both aptamer and catalytic nucleic acid sequences (ribozymes or DNAzymes). Binding of a ligand to the aptamer induces a conformation change in the aptazyme which activates a ribozyme or DNAzyme.
Sando and colleagues (2003) developed a signal amplification strategy that used sensing molecules (target-assisted self cleavage (TASC) probes), which contained multiple domains constituting a target sensing sequence, a DNAzyme domain and a dual labelled, DNA/RNA chimeric substrate for the adjoined DNAzyme. While this method avoids the use of protein enzymes, the TASC probes are complex and expensive molecules which must be custom made for each new target.
Several groups have reported the detection of nucleic acid targets, and other analytes with colourimetric readouts (Elghanian et al., 1997, Mirkin et al, 1996, and Liu and Lu, 2004). The strategy uses nanoscopic gold particles tagged with oligonucleotides. The gold particles can then be aggregated by the addition of a “bridging oligonucleotide”, causing a change in colour from red to blue (Mirkin et al, 1996). Liu and Lu (2004) extended this strategy by incorporating a DNAzyme substrate into the bridging oligonucleotide, such that activation of the DNAzyme results in cleavage, dispersal of the gold particles and a change in colour from blue to red. The group used the approach to detect lead using a lead sensitive DNAzyme, and to detect adenosine using an aptazyme.
Several examples of amplification cascades, which use catalytic nucleic acids, are known in the art. The zymogene/DzyNA approach combines target amplification (e.g. PCR), with DNAzyme replication. The DNAzymes, which are co-amplified with the target, cleave one or more universal reporter substrate (s) permitting generic detection of one or more targets (U.S. Pat. No. 6,140,055; U.S. Pat. No. 6,201,113; U.S. Pat. No. 6,365,724; WO 99/45146, Todd et al., 2000). Strategies for amplification cascades have been devised which use catalytic nucleic acids, instead of protein enzymes to mediate amplification. In another approach, a signal amplification cascade used two inactive, circularized 10:23 DNAzymes which were capable of activating each other by cross cleavage resulting in linearisation. Paul and Joyce (2004) described a replication cascade mediated by a ribozyme with ligase activity. In this reaction, a first ribozyme ligates two RNA containing oligonucleotides to form a second ribozyme. The second ribozyme then ligates two other RNA containing oligonucleotides to form a new first ribozyme, thus triggering a replication cascade, which produces new copies of both the first and second ribozyme.
Nucleic acid cascades have been considered for a range of biotechnological applications, especially in diagnostics. They could allow detection of proteins and nucleic acids for disease diagnosis by facilitating signal amplification. Catalytic nucleic acids and/or cascade reactions can be used for applications other than diagnostics, for example, within the field of computation analysis and biomolecular engineering of nano-scale devices and switches which may be used in therapeutics.
Devices that can convert information from one form into another, according to a finite procedure, are called automata. A programmable finite automaton, which was capable of solving computational problems was developed using protein enzymes (a restriction endonuclease and a ligase) and double stranded DNA (Benenson et al, 2001). The enzymes serve as the “hardware” and the DNA encodes the “software”. The input and automata are programmed by selection of the appropriate DNA software. The automaton proceeds via a cascade of cleavage, hybridization and ligation cycles, producing a detectable output molecule that encodes the automaton's final state and thus the computational result.
Simple molecular-scale programmable computers, which use biological molecules as input data and biologically active molecules as outputs, could be used to create systems for the logical control of biological processes (Benenson et al, 2004). As proof of concept in vitro, Benenson et al developed a bio-molecular computer that was capable of (i) measuring the abundance of specific messenger RNA species, and (ii) responding by releasing a single stranded DNA anti-sense molecule capable of affecting gene expression. Another molecular automaton, which used a network of DNAzymes to create molecular-scale logic gates, was programmed to play “tic-tac-toe” (Stojanovic and Stefanovic, 2003). Recently, a binary DNAzyme with ligase activity was engineered to recognize and hybridize (“read”) to one sequence, and to ligate (“write”) a separate, distinct sequence, which in turn could be amplified by PCR (Tabor et al, 2006).
Methods where DNA computation interfaces with biology can have broad applications. For example, a simple DNA logic gate could regulate release of insulin based on a combination of physiological signals, for example, high blood sugar and low glucagon (Cox and Ellington 2001). Together, the progress in discovering new functionality for nucleic acids has provided a series of tools, such as aptamers and catalytic nucleic acids, and new structural components which allow the development of components for new nano-scale molecular “machines”.
Several processes have been used to operate nanodevices and automata including (i) hybridization processes, which include branch chain migration, inhibition of hybridisation between complementary strands by secondary structure formation, for example hairpin formations, (ii) cleavage using a restriction endonuclease and (iii) induction of conformation changes such as rotation around a central DNA axis, shrinking/extension and translatory movements. A modular DNA signal translator for the controlled release of a protein by an aptamer used an arbitrary DNA sequence as “input” (Beyer and Simmel, 2006).
Thus, there is an ongoing need in the art for simple, fast, and cost effective methods for the detection of targets, and for assembly of nano-scale devices, including programmable devices, which can be performed using stable, nucleic acid components.